Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. The parasite has a complex secretory system essential for host cell invasion and establishment of the parasitophorous vacuole. Biogenesis of these secretory organeles remains poorly understood but several groups have postulated a compartment situated between Golgi and mature micronemes and rhoptries in which sorting and processing occurs. We have recently discovered a novel post-Golgi compartment of T. gondii that we named the plant-like vacuole (PLV) and the goal of this project is to investigate its function in the biology of the parasite. This organelle is a large multi-vesicular structure that possesses similarities to the central vacuole found in plant cells. This organelle can be labeled with antibodies against proteins with great similarity to vacuolar plant pumps and channels, such as a K+sensitive V-H+PPase (TgVP1), and a tonoplast-like aquaporin (TgAQP1). Physiological studies further revealed similarities to plant vacuoles such as the presence of a V-H+ATPase, Na? and Ca2? antiporters, and calcium storage. Furthermore, as plant vacuoles, the PLV also contains proteases such as cathepsin L (TgCPL) and others. Our results and those of our collaborators showed that the PLV is a prominent feature of extracellular tachyzoites and, as the plant vacuole, has multiple potential functions including roles in protein sorting and resistance to environmental stresses. For example, the role of this vacuole in the proteolytic maturation of at least two microneme precursor proteins, proTgM2AP and proTgMIC3, suggests that this organelle could represent an endocytic/exocytic hub where sorting of proteins targeted to various organelles occurs. T. gondii tachyzoites that overexpress VP1 in the PLV, become more resistant to sodium stress pointing to an important role of the PLV in resistance to environmental ionic stress. Ionic stress is a major challenge that T. gondii confronts when exiting the host cell into the extracellular media. There is a dramatic change in the concentration of sodium, potassium, calcium and other ions between the intracellular and the extracellular environment. Dealing with these stressful changes is important for the survival of the parasite, which needs to actively invade other host cells to continue with its lytic cycle. We will test two hypotheses, which have been developed based on the functions of the plant vacuole and preliminary observations in the parasite, using a combination of genetic and cell biological experiments. This novel organelle acts to regulate ions and/or as a post Golgi sorting compartment for secretory proteins destined to the micronemes, rhoptries, and acidocalcisomes. These models are not mutually exclusive if we take into account the fact that the plant vacuole is home to a complex set of functions (storage, sorting, stress, plant growth, etc.). We think that the PLV plays a central role during the extracellular phase of the parasite not only in resisting environmental stress but also as it prepares itself for invading the next host cell. PUBLIC HEALTH RELEVANCE: This work is designed to test the hypotheses that the plant-like vacuole (PLV) of T. gondii is involved in the regulation of calcium and pH homeostasis and response to salt stress, as well as in sorting and processing of proteins to organelles such as the rhoptries and acidocalcisomes. Our work could provide potential targets for chemotherapy.